1. Field of the Invention
The present invention concerns non-invasive methods for detecting the onset, progression and regression of gynecologic cancers. More particularly, the present invention relates to the detection of human chorionic gonadotropin beta-subunit core fragment as a marker of gynecologic cancers.
2. Background Information
Cervical cancer is one of the most common malignancies afflicting women (E. Silverberg, "Cancer Statistics", CA-A Cancer J. Clinicians, 6, 9-26, (1986)). The Pap (Papanicolaou) smear has led to early diagnosis and has mainly been responsible for the overall improvement in survival reported for this disease (Yajima, A., Mori, T., Sato, S , Wakisaka, T , and Suzuki, M., "Effect of Cytologic Screening on the Detection of Cervical Carcinoma", Obstet. Gynecol., 59, 565-568 (1982). However, patients who are not able or do not wish to undergo Pap smear (an invasive method) screening run the risk of unsuccessful treatment if they wait for symptoms (pain, bleeding and/or discharge) to develop. This often is the situation with women who are in epidemiologic groups at highest risk for developing cervical cancer. For example, the National Cancer Institute of Peru recently reported that 80% of 1,100 new cervix cancer patients seen annually have stage III or IV disease. Routine Pap Smear screening is not accepted by these patients and the survival is extremely poor (Castellano, C., "Manejo Del Paciente Con Citologia Anormal", Ninth Congress, Cancer in Peru, Lima, (1985)).
The Pap smear as a screening technique for cervical cancers, however, can be inaccurate. False negative rates for Pap smears vary greatly from 12.5% to 45% (C. Castellano, supra; Jordan, S. W., Smith N. L., and Dike, L. S., "The Significance of Cervical Cytologic Dysplasia", Acta Cytol., 25, 237-244, (1981)) .
Among American women, the ovary is the second most common site of gynecologic cancer (E. Silverberg, supra). The common epithelial ovarian cancers lack early warning symptoms and there are no routine tests, like the Pap smear, for early detection. Ovarian cancer is usually not suspected until a pelvic mass is present and, if not detected until advanced stage, is almost always fatal (Schwartz, P. E., "Gynecologic Cancer" In: J. A., Spittle, Jr. (ed.), Clinical Medicine, pp. 1-44. Philadelphia: Harper and Row, (1985)).
A readily available urine test that would aid in the detection of women at increased risk for having gynecologic malignancies, particularly cervix and ovarian cancer would be a major step forward for patients in whom advanced stage disease is almost always fatal. Such a test may obviate the need among those with religious or ethical concerns for an unacceptable pelvic examination and Pap smear.
The efficacy of treatment for patients with recurrent gynecologic cancer is reflected in the volume of cancer at the time recurrence is documented and the sites of recurrent disease. Therapy may be of limited value when recurrent disease is not identified until the patient has clinical signs or symptoms. Early recognition of occult persistent or recurrent cancer may lead to more effective therapeutic intervention.
Surgical intervention in the form of radical surgery may cure patients with central recurrence of cervical cancer. Diagnosis of persistent or recurrent central disease in a radiation field may be difficult to confirm by cytologic or biopsy techniques. An accurate tumor marker for cervical cancer may lead to earlier recognition and more rapid diagnosis and treatment.
Previous experience suggested a role for lipid-associated sialic acid (LASA) in this regard, but not for squamous cell carcinoma antigen (SCC) (Schwartz, P. E., Foemmel, R. S., Chambers, S. K., and Chambers, J. T., "Evaluation of Squamous Cell Carcinoma Antigen (SCC) and Lipid-association Sialic Acid (LSA) in Monitoring Patients With Cervical Cancer", Proc. Am. Soc. Clin. Oncol., 6, 113 (1987)).
Early recognition of recurrent or persistent endometrial cancer may lead to more rapid treatment with potentially more effective combination chemotherapy (Seski, J. C., Kasper, G. L., and Kunschner, A. J., "Chemotherapy for Endometrial Cancer, in Diagnosis and Treatment Strategies" (F. N. Rutledge, R. S. Freedman and D. M. Gershenson Eds.), University of Texas Press, Austin, pp. 327-334 (1987)).
Multiple circulating markers have been evaluated in the management of epithelial ovarian cancer patients, the most promising of which is CA 125 (Bast, R. C., Kung, T. L., St. John, E., Jenison, E., Niloff, J. M., Lazarus, H., Berkowitz, R., Leavitt, T., Griffiths, T., Parker, L., Zurawski, V. R., and Knapp, R. C., "A Radioimmunoassay Using a Monoclonal Antibody to Monitor the Course of Epithelial Ovarian Cancer", N. Engl. J. Med., 309, 883-887 (1983)).
As high as eighty percent of patients with non-mucinous ovarian cancers can have elevated levels of CA 125 in their serum, which will become nondetectable as the cancer responds to treatment. Unfortunately, in a recently reported clinical trial using CA 125 in the management of ovarian cancer, 6 of 11 (55%) patients who were clinically free of disease and had CA 125 levels that were in the normal range, were found to have persistent cancer at a second-look procedure (Atack, D. B., Nisker, J. A., Allen, H. H., Tustanoff, E. R., and Levin, L., "CA 125 Surveillance and Second-look Laparotomy in Ovarian Carcinoma", Am. J. Obstet. Gynecol., 154, 287-289 (1986)).
Similarly, at Yale-New Haven Hospital the published false-negative rate for CA 125 (cut-off 35 U/ml) at second-look surgery is 40% (Schwartz, P. E., Chambers, S. K., Chambers, J. T., Gutmann, J., Katopodis, N., and Foemmel, R. S., "Circulating Tumor Markers in the Monitoring of Gynecologic Malignancies", Cancer 60, 353-361 (1987) Other early clinical trials have shown that decreasing CA 125 levels in ovarian cancer patients are not necessarily an indicator of regressive disease (Alvarez, R. D., To, A., Boots, L. R., Shingleton, H. M., Hatch, K. D., Hubbard, J., Soong, S. J., and Potter, M. E., "CA 125 as a Serum Marker for Poor Prognosis in Ovarian Malignancies", Gynecol. Oncol., 26, 284-309, (1987)).
Once the diagnosis of ovarian cancer is established the currently available clinical markers tend to parallel clinical examination findings, but do not guarantee the efficacy of the treatment in those patients who are clinically free of disease, nor are they sufficiently sensitive to avoid the use of second-look operations.
Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of the following two dissimilar subunits: alpha 92 amino acids long and beta 145 amino acids long, joined non-covalently. It is normally produced by trophoblast tissue and can be detected in the blood and urine of women in pregnancy or trophoblast disease. Free forms of hCG alpha and beta subunits, which can account for as much as 90% of the total produced, are also found in blood and urine in pregnancy and trophoblast disease (Cole, L. A., Kroll, T. G., Ruddon, R. W., and Hussa, R. O., "Differential Occurrence of Free B and Free A Subunits of Human Chorionic Gonadotropin in Pregnancy Sera", J. Clin Endocrinol. Metab., 58, 1200-1202 (1984); Cole, L. A., "Occurrence and Properties of Glycoprotein Hormone Free Subunits", in Microheterogeneity of Glycoprotein Hormones (H. Grotjan and B. Keel, Eds.), CRC Press, New York, in press; Cole, L. A., Hartle, R. J., Laferla, J. J., and Ruddon, R. W., "Detection of the Free beta-subunit of Human Chorionic Gonadotropin In Cultures of Normal and Malignant Trophoblast Cells, Pregnancy Sera, and Sera of Patients with Choriocarcinoma", Endocrinology, 113, 1176-1178 (1983)
Numerous researchers have shown that human chorionic gonadotropin is present in the circulation of approximately 20% of women with cancer. Because of the low percentage positive for cancer it has not been used as a marker for gynecological cancer.
Free beta-subunit missing sialic acid residues on its carbohydrate moieties (asialo free beta), and fragmented asialo free beta (core fragment) composed of residues 6-40 disulfide-linked to 55-92, are also produced in pregnancy and trophoblast disease (Birken, S. , Armstrong, E. G., Kolks, M. A. G., Cole, L. A., Agosto, G. M., Krichevsky, A., and Canfield, R. E., "The Structure of the Human Chorionic Gonadotropin beta Core Fragment from Pregnancy Urine", Endocrinology, in press.) .
Free beta-subunit, asialo free beta and the core fragment of asialo beta-subunit, together called UGF, are rapidly cleared from the circulation and are more-readily detected in urine than in serum samples (Schroeder, H. R., and Halter, C. M., "Specificity of Human beta-Choriogonadotropin Assays for the Hormone and for an Immunoreactive Fragment Present in Urine During Normal Pregnancy", Clin. Chem., 29, 667-671 (1983); Lefort, G. P., Stolk, J. M., and Nisula, B. C., "Renal Metobolism of the beta-subunit of Human Choriogonadotropin in the Rat", Endocrinology, 119, 924-931 (1986); Wehmann, R. E., and Nisula, B. C., "Metobolic Clearance Rates of the Subunits of Human Chorionic Gonadotropin in Man", J. Clin. Endocrinol. Metab., 48, 753-759 (1979)) .
Ectopic hCG has been detected in the blood and tissues of patients with non-trophoblastic cancers, most notably gynecologic malignancies (R. O. Hussa, "Human Chorionic Gonadotropin, a Clinical Marker: Review of its Biosynthesis", Ligand Rev., 3, 1-43, (1981))
In a recent compilation of 38 separate studies (n=692), 36% of women with cervical, 27% of those with endometrial and 13% of those with vulvar cancers were shown to have detectable levels of hCG in radioimmunoassays. This low percentage with detectable levels and the associated low titers have, however, restricted the use of hCG in detecting and following the therapy of gynecologic cancers (Hussa, supra). In studies of women with gynecologic cancer, using the Hybritech "Tandem" hCG-specific immunoradiometric assay (&lt;0.1% hLH and free subunit crossreactivity), it was found that only 11 of 64 (17%) had detectable (&gt;0.2 ng/ml, equivalent of &gt;2.0 mIU/ml) serum levels of hCG (Wang, Y., Schwartz, P. E., Cole, L. A., "Serum hCG Investigation in Patients with Non-trophoblastic Cancer", J. Obstet. Gynecol. China, in press). The average level was found to be just 0.30 ng/ml (equivalent of 3 mIU/ml). Serial serum samples from 14 cancer patients with elevated hCG levels were examined. Levels were measured at the start and following therapy. Strangely, in 8 of 10 women with progressive cancer and increasing tumor mass hCG levels went down (Wang et al, supra). Furthermore, in 4 of 4 women with regressing disease and diminution of tumor mass, hCG levels went up. Clearly, serum hCG has very limited value in screening and in the management of patients with gynecologic cancer.
hCG free beta-subunit and core fragment, UGF, have also been detected in patients with non-trophoblastic cancers (Papapetrou, P. D., and Nicopoulou, S. C., "The Origin of a Human Chorionic Gonadotropin beta-subunit Core Fragment in the Urine of Patients with Cancer", a Endocrinologica, 112, 415-422 (1986); Vaitukaitis, J. L., "Characterization of a Small Molecular Size Urinary Immunoreactive Human Chorionic Gonadotropin (hCG)-like Substance Produced by Normal Placenta and by hCG-secreting Neoplasms", J. Clin. Endocrinol. Metab. , 53, 1014-20 (1981)) .
In patients with UGF in urine, the fact that it originates from the cancer tissue itself has been established by the finding of significant levels (average 1.0 ng/mg protein) in 5 of 5 tumor tissue homogenates. In a preliminary study of UGF in patient urines (Cole, L. A., Wang, Y., Elliott, M., Latif, M., Chambers, J. T., Chambers, S. K., and Schwartz, P. E., "Urinary Human Chorionic Gonadotropin Free beta-Subunit and beta-Core Fragment: A New Marker of Gynecologic Cancers, Cancer Res., 48, 1356-1360, (1988)) levels were measured in spot samples from 50 healthy women and from 68 patients with active gynecologic cancer. Elevated levels (&gt;0.2 ng/ml beta-subunit, or molar equivalent of the beta core fragment) were detected in 3 of the control and 50 of the cancer samples. Although adjustments were not made for urine concentration (creatinine level), a sensitivity of 74 % and a specificity of 94% of UGF for gynecologic cancers was suggested. These preliminary findings showed that gynecologic cancers more commonly produce free-subunits or beta core fragment, than hCG, and that the use of UGF as a tumor marker warranted further investigation.